G protein-coupled receptors (GPCRs) are a large family of cell surface receptors that regulate a multitude of biological processes in response to a diverse array of stimuli, and they are important drug targets. The class B/Secretin family of GPCRs in humans includes fifteen receptors that are activated by diverse neuropeptides, peptide paracrine factors and peptide endocrine hormones. Class B GPCRs comprise an extracellular domain (ECD) of about 120 amino acids in addition to a 7-transmembrane (7TM) domain in the membrane. The ECD has an N-terminal a-helix and a set of β-sheets held together by three disulfide bonds. Activating peptides bind class B GPCRs via a “two-domain” model, whereby the C-terminal region of the peptide binds the ECD, and the N-terminal region of the peptide binds and activates the 7TM domain. Certain of the activating peptides bind as extended a-helices to the same region of the receptor, in a groove between the N- and C-termini of the isolated ECDs. However, the mechanism of binding of the calcitonin (CT) family of this group of Class B GPCR-activating peptides has been unknown.
Members of the CT family of activating peptides include, for example, calcitonin gene-related peptide alpha (αCGRP), calcitonin gene-related peptide beta (βCGRP), adrenomedullin (AM), adrenomedullin 2/intermedin (AM2), amylin (Amy), and CT. These C-terminally amidated peptides have a range of actions including neurogenic inflammation, e.g., as a factor in migraine headache pathogenesis (CGRP), vasodilation/cardioprotection (CGRP, AM and AM2), and regulation of blood and lymphatic vascular development (AM), nutrient intake and blood glucose (Amy), and bone turnover (CT). CGRP in the trigeminovascular system acts as a pain signaling neurotransmitter. Elevated CGRP levels are associated with migraines, and exogenous administration of CGRP to migraineurs triggers migraines. Small molecule antagonists of the CGRP receptor that bind the ECD complex showed efficacy in clinical trials, but were abandoned because of toxicity unrelated to CGRP signaling.
Binding of CGRP, AM, and AM2 to their cognate class B receptor, the calcitonin receptor-like receptor (CLR), is dependent on association of CLR with one of three accessory membrane proteins that determine ligand selectivity: receptor activity-modifying proteins (RAMPs) 1, 2, or 3. RAMPs have an ECD of about 100 amino acids and a single TM segment. CLR:RAMP1 is a CGRP receptor, CLR:RAMP2 preferentially recognizes AM and is called the AM1 receptor, and CLR:RAMP3 binds both AM and AM2 with high affinities and is called the AM2 receptor. Amy has a low affinity for the class B CT receptor (CTR); however, when CTR associates with any of the RAMPs, its affinity for Amy is markedly increased. CTR alone is the receptor for CT. Thus the RAMPs profoundly alter the behavior of CLR and CTR.
For peptide-based therapeutics targeting the CGRP or AM receptors, selectivity against the CTR and AMY receptors is desired because of their different functions. CTR is involved in calcium homeostasis and bone remodeling. Salmon CT, which has higher affinity for human CTR than human CT, has long been used as a therapeutic for Paget's disease and osteoporosis. Amy signaling controls blood glucose levels and regulates food intake. The Amy analog Pramlintide is used as an insulin adjunct therapy for types I and II diabetes, and AMY activation is explored as an obesity treatment.
Crystal structures have been available for ligand-free and small molecule antagonist-bound CLR:RAMP1 and ligand-free CLR:RAMP2 ECD complexes, but these have provided little insight into how the peptides bind or how RAMPs determine selectivity. In a further complication, it has appeared unlikely that CGRP and AM bind as extended helices as seen with other class B peptide ligands; there is evidence that only a small portion of these peptides form α-helices and that at their C-termini, there are one or more turn structures. A better understanding of the mechanism of binding of CGRP and AM to their respective CLR:RAMP receptor complexes has been desired.